Malaysian cockle (Anadara granosa) allergy: Identification of IgE-binding proteins and effects of different cooking methods

The purpose of this study was to evaluate the effect of different cooking methods on the allergenicity of cockle and to identify proteins most frequently bound by IgE antibodies using a proteomics approach. Raw, boiled, fried and roasted extracts of the cockle were prepared. The protein profiles of the extracts were obtained by separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis (2-DE). IgE-immunoblotting was then performed with the use of individual sera from patients with cockle allergy and the major IgE-binding proteins were analyzed by mass-spectrometry. SDS-PAGE of raw extract showed 13 protein bands. Smaller numbers of protein bands were detected in the boiled, fried and roasted extracts. The 2-DE gel profile of the raw extract further separated the protein bands to ~50 protein spots with molecular masses between 13 to 180 kDa and isoelectric point (pI) values ranging from 3 to 10. Immunoblotting of raw extract exhibited 11 IgE-binding proteins with two proteins of 36 and 40 kDa as the major IgE-binding proteins, while the boiled extract revealed 3 IgE-binding proteins. Fried and roasted extracts only showed a single IgE-binding protein at 36 kDa. 2-DE immunoblotting of raw extract demonstrated 5 to 20 IgE reactive spots. Mass spectrometry analysis led to identification of 2 important allergens, tropomyosin (36 kDa) and arginine kinase (40 kDa). Heated extracts showed a reduction in the number of IgE-reactive bands compared with raw extract, which suggest that thermal treatment can be used as a tool in attempting to reduce cockle allergenicity. The degree of allergenicity of cockle was demonstrated in the order raw > boiled > fried ≈ roasted. Two important allergens reacting with more than 50% of patients’ sera identified using mass spectrometric approaches were tropomyosin and arginine kinase. Thus, allergens found in this study would help in componentbased diagnosis, management of cockle allergic patients and to the standardisation of allergenic test products as tools in molecular allergology.

Identification of Major and Minor Allergens of Mud Crab (Scylla Serrata)

Crab meat is a valuable source of proteins and functional lipids and it is widely consumed worldwide. However, the prevalence of crab allergy has increased over the past few years. In order to understand crab allergy better, it is necessary to identify crab allergens. The aim of the present study was to compare the IgEbinding proteins of raw and cooked extracts of mud crab (Scylla serrata). Raw and cooked extracts of the mud crab were prepared. Protein profiles and IgE reactivity patterns were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting using sera from 21 skin prick test (SPT) positive patients. In SDS-PAGE, 20 protein bands (12 to 250 kDa) were observed in the raw extract while the cooked extract demonstrated fewer bands. Protein bands between 40 to 250 kDa were sensitive to heat denaturation and no longer observed in the cooked extract. In immunoblotting experiments, raw and cooked extracts demonstrated 11 and 4 IgE-binding proteins, respectively, with molecular weights of between 23 and 250 kDa. A heat-resistant 36 kDa protein, corresponding to crab tropomyosin was identified as the major allergen of both extracts. In addition, a 41 kDa heat-sensitive protein believed to be arginine kinase was shown to be a major allergen of the raw extract. Other minor allergens were also observed at various molecular weights.